Inhibitors of the inositol polyphosphate 5-phosphatase SHIP2 molecule

ABSTRACT

The present invention is related to an inhibitor of the inositol polyphosphate 5-phosphatase SHIP2 protein or its encoding nucleotide sequence identified by SEQ ID NO. 1 or of SHIP2 mRNA expression.  
     The present invention is also related to a pharmaceutical composition comprising said inhibitor and an adequate pharmaceutically acceptable carrier or diluent and to a non-human knock-out mammal comprising homozygously or heterozygously a partial or total deletion in its genome of the inositol polyphoshate 5-phosphatase SHIP2 nucleotide sequence.

FIELD OF THE INVENTION

[0001] The present invention relates generally to the fields ofdiabetes. More particularly, it concerns the identification of genes andproteins responsible for diabetes and to their inhibitors for use intherapeutics.

BACKGROUND OF THE INVENTION AND STATE OF THE ART

[0002] Insulin is the primary hormone involved in glucose homeostasis.Partial or total deficiency in insulin secretion or action leads toimpaired glucose metabolism and diabetes.

[0003] Diabetes is a major cause of health difficulties in the world.Non-insulin dependent diabetes mellitus (NIDDM also referred to as Type2 diabetes) is a major public health disorder of glucose homeostasisaffecting about 5% of the general population in the United States. Thecauses of the fasting hyperglycemia and/or glucose intoleranceassociated with this form of diabetes are not well understood.

[0004] Clinically, NIDDM is a heterogeneous disorder characterised bychronic hyperglycemia leading to progressive micro- and macro-vascularlesions in the cardiovascular, renal and visual systems as well asdiabetic neuropathy. For these reasons, the disease may be associatedwith early morbidity and mortality.

[0005] In the field of genomics, various mutations in the diabetessusceptibility genes were identified, for instance in the hepatocytenucleotide factor genes family (HNF-1, HNF-4 and HNF-6) as described indocuments WO98/11254 and WO98/23780.

[0006] The role of said genes in biochemical pathways affectingsynthesis or secretion of insulin by the beta cells of Langerhans isletshas been identified by the phenotype analysis of knock-out mice whereinsaid genes or portions thereof have been deleted from their genome.

[0007] Said knock-out mammal are thereafter used as models for theidentification of new compounds or new methods of treatment which couldbe used for decreasing the symptoms resulting from diabetes.

[0008] It is also possible to use the corresponding identified geneswhich will be present in a sufficient amount in a pharmaceuticalcomposition for the treatment and/or the prevention of said disease.

[0009] However, in this field, it exists a need for the identificationof new target and biological pathways which could be used for improvingthe treatment and/or the prevention of diabetes.

[0010] Type 2 SH2-domain-containing inositol polyphosphate 5-phosphataseor SHIP2 is closely linked to phosphatidylinositol 3′-kinase andShc/ras/MAP kinase-mediated signaling events in response to stimulationby specific growth factors.

[0011] The structure of SH2-domain containing enzymes and presenting aphosphatase catalytic activity has been already described by Pesesse etal. (1997 and 1998) and Erneux et al. (1998).

[0012] It is known that said SH2-domain containing proteins showssimilarity with another known inositol polyphosphate 5-phosphataseidentified as molecule SHIP1 and shows also 99% identity to a previouslyreported sequence (INPPL-1). INPPL-1 however, did not contain an SH2domain.

[0013] Said new sequence will be identified hereafter as the moleculeSHIP2. The other known inositol 5-phosphatase SHIP1 has been the subjectof an intensive research because it may be possibly involved in negativeSignalling of B immune cells and could be therefore used as target forthe screening of new molecules having possibly therapeutical and/orprophylactic properties in the treatment of various immune inflammatoryor allergic symptoms and diseases.

[0014] Toshiyasusasaoka et al. (Diabetes Vol. 84, No. PPA 60 (1999)(XP000905226)) describe the cloning characterisation of a rat SHIP2molecule that does not have the SAM domain present in human SHIP2. Theyshow that overexpression of the SHIP2 molecule inhibits insulin-inducedPKB activation by the 5-inositol phosphatase activity of SHIP2. Theauthors suggest that SHIP2 plays a negative regulatory role in diversebiological action of insulin and that the dual regulation of theSHC-Grb2 complex and downstream molecule of PI3-kinase provides possiblemechanisms of SHIP2 molecule to participate in insulin signalling.

[0015] The international patent application WO 97/12039 describes thepurification and the isolation of the nucleic acid molecules comprisinga sequence encoding the SH2-containing inositol-phosphatase, a vectorcomprising said sequence, a cell transformed by said vector and apurified and isolated SH2-containing inositol-phosphatase moleculeexpressed by said cell. This document describes also antibodies directedagainst said protein and a method for identifying a substance capable ofbinding to said protein.

[0016] However, the precise functions of said SH2-domain containinginositol polyphosphate 5-phosphatase SHIP2 has not yet been identified.

SUMMARY OF THE INVENTION

[0017] The inventors have discovered unexpectedly that a knock-outmammal, preferably a knock-out mouse, comprising the partial or totaldeletion (heterozygously or homozygously) of said SH2-domain-containingpolyphosphate inositol 5-phosphatase SHIP2 sequence is hypersensitive toinsulin (severe postnatal hypoglycaemia and deregulated expression ofgenes involved in gluconeogenesis).

[0018] This increased sensitivity is so important that after two orthree days following the birth, the homozygote (SHIP2−/−) mice die fromsevere hypoglycemia, and that an unpaired glucose tolerance is observedin heterozygote (SHIP2+/−) mice.

[0019] In vitro, the absence of one or both normal alleles of the SHIP2gene is associated with an increased activation of PKB, an effector ofPdtIns 3-kinase cascade, and of MAP kinase in response to insulin. Theseresults provide the first direct evidence that SHIP2 is a potentnegative regulator of insulin signalling in vivo, and a potentialtherapeutic target for the treatment of type II diabetes.

[0020] A first aspect of the present invention is related to aninhibitor of said inositol polyphosphate 5-phosphatase SHIP2 molecule,preferably a human molecule, said inhibitor being directed against saidmolecule and being able to reduce or block its activity or expression.

[0021] A first preferred example of said inhibitor is an anti-sense RNA(of 8 to 50 bases, preferably from 10 to 30 bases in length) constructedfrom the complementary sequence of the messenger RNA that can be deducedfrom the sequence of SHIP2 molecule complementary DNA encoding thesequence (identified hereafter as SEQ ID NO. 1 and by Emeux et al.(1998)) or its complementary strand and which specifically hybridisesand inhibits its expression. Said inhibitors can also be a molecule thatdirectly or indirectly decreases SHIP2 mRNA expression (transcriptionfactors) or stability.

[0022] A second example of said inhibitor is a mutated SHIP2 molecule ora portion thereof of more than about 30, about 50, about 100 or about150 amino-acids (negative dominant) that would prevent the naturalactivity of SHIP2 by competition of the mutated molecule to interactingproteins or receptors involved in insulin production cascade.Preferably, said mutated SHIP2 molecule or portion comprises a mutationin the following amino acid sequence: RTNVPSWCDR, especially one or moremutations, preferably of the following amino acids: S, C, N, D or R ofsaid specific SHIP2 portion sequence. Said mutation(s) affect(s) thecatalytic site of the SHIP2 molecule (phosphatase activity), asdescribed in Emeux et al. (1998). Those mutations typically would createdominant negative effects.

[0023] Other examples of said inhibitors are substrates of said SHIP2molecule (being an enzyme) and analogues of its substrates such as thephosphatidylinositol 3,4,5-triphosphate, the inositol1,3,4,5-tetrakisphosphate, the inositol 1,4,5 triphosphate adenophostinor any available analogue of the inositol phosphate structure includingthe membrane-permeant esters or phosphatidylinositol 3,4,5-triphosphatethat have been used to deliver the phosphatidylinositol3,4,5-triphosphate across cell membranes.

[0024] Said inhibitor can be also a competitive inhibitor, such as the2,3-biphosphoglycerate, thiol blocking agents or any protein phosphataseinhibitor such as the okadaic acid or the orthovanadate. The inhibitorcan be also a specific portion of more than about 30, about 50, about100 or about 150 amino-acids of the protein structure of said enzyme,preferably a portion comprising the SH2-domain (the first 110 aminoacids of the sequence SEQ ID NO. 1, the proline-rich domain (the last351 amino acids of the sequence SEQ ID NO. 1) or more preferred portionsof said sequence which are able to compete with the molecule SHIP2recruitment to plasma membranes, thereby inhibiting its activity. Theterm “competitive inhibitor” is well accepted in the art.

[0025] The inhibitor could also reduce or block the activity of theSHIP2 molecule, or reduces the expression of the SHIP2 mRNA.

[0026] The inhibitor or a pharmaceutical composition comprising anacceptable carrier or diluent or the inhibitor according to theinvention, being able to reduce or block the activity of the SHIP2molecule, increases the sensibility of a patient (including a human) toinsulin and could be advantageously used in the treatment or theprevention of type II diabetes and associated diseases.

[0027] Another aspect of the present invention is related to a method,kit or apparatus for the detection of known or unknown compounds whichcould be used as inhibitors of said inositol polyphosphate 5-phosphataseSHIP2 molecule. Said method comprises the steps of submitting saidunknown compounds to an assay based upon the analysis of SHIP2 moleculeactivity. Said assay, is advantageously performed on a molecularconstruct containing the catalytic domain of the SHIP2 enzyme (for thecorresponding human SHIP2 molecule identified in the enclosed sequenceSEQ ID NO. 1, this ranges from the amino acid 427 to the amino acid 729or one or more specific portions of said catalytic domain), saidconstruct being expressed in a micro-organism, preferably in E. coli, orin a cell line and the assay comprising the means for quantifying thehydrolysis of inositol 1,3,4,5-tetrakisphosphate and the production ofinositol 1,3,4-triphosphate. The expression of SHIP2 in bacteria E. colihas been described in Pesesse et al., 1998.

[0028] The kit, apparatus and method according to the invention comprisesubstrates commercially available. The assay according to the inventioncould also be based upon a quantification of the hydrolysis ofphosphatidylinositol 3,4,5-triphosphate followed by thin layerchromatography analysis. A decrease in the amount of a hydrolysisproduct of 5-phosphatase activity refers to 10% or greater decrease in asignal analysed by the thin layer chromatography in the presence of aninhibitor relative to its absence.

[0029] Another analysis of possible inhibitors of the SHIP2 moleculeactivity is based upon the inhibition of SHIP2 phosphorylation ontyrosine, because this index of activation has been shown to be crucialin the activation process and the insulin sensitive pathways. The SHIP2molecule is purified and tested for its phosphorylation by Western blotanalysis with anti-phosphotyrosine antibody which could be used also asan inhibitor according to the invention. Another analysis of possibleinhibitors of SHIP2 molecules is based upon the reduction or inhibitionof SHIP2 mRNA expression, because reduced expression of SHIP2 mRNA invivo is associated with increased isulin sensitivity. SHIP2 promoterregion or untranslated regions (5′ or 3′) of SHIP2 cDNA are linked witha reporter gene (luciferase, CAT, . . . ) in a plasmid vector andtransfected into cells in culture. Compounds to be tested are added ornot to the cells, and reporter gene activity is recorded to fond outcompounds that reduce reporter gene activity, i.e. compounds that actthrought the promoter, 5′ or 3′ untranslated regions of SHIP2 sequencesto reduce the production of the protein. A reduction of expressionrefers to a 10% or greater decrease in SHIP2 mRNA level in the presenceof an inhibitor relative to its absence.

[0030] A last aspect of the present invention is related to a non-humanknock-out mammal comprising (homozygously or heterozygously) a partialor total deletion (in its genome) of the genetic sequence encoding theinositol polyphosphate 5-phosphatase SHIP2 enzyme, said knock-out mammalbeing hypersensitive to insulin and which could be used as a model forthe study of diseases like severe hypoglycaemia in human newborn, orunpaired glucose tolerance in adult humans as well as the study of theirtreatment. Said genetic sequence encodes the inositol polyphosphate5-phosphatase SHIP2 molecule (enzyme) identified hereafter as SEQ ID NO.1 and described by Pesesse et al. (1997).

[0031] Said non-human knock-out mammal is preferably a knock-out mouse,obtained by techniques well-known by a person skilled in the art. Saidmammal is preferably obtained by a genetic modification, a partial ortotal deletion in the wild type sequence through the integration of aforeigner nucleic acid sequence. Said genetically modified sequence isincorporated in a vector or electroporated and reintroduced in anembryonic stem cell (ES) for which cellular clones are selected beforethe integration preferably in a Swiss pseudo-gravide morula-embryoaccording to the technique described by Carmeliet et al. (1996),allowing thereafter the selection of mice comprising the heterozygousmodification of said wild-type sequence and after crossing homozygouslygenetically modified mice.

BRIEF DESCRIPTION OF THE DRAWINGS

[0032]FIG. 1 represents the targeted disruption of SHIP2 gene. (a, Thewild type allele (top) and the targeting vector (bottom) are represented(exons, closed boxes), as well as the 2 probes used in Southern analysisand the DNA fragments generated after digestion with EcoRI and BamHI. R:EcoRI; B: BamHI. b, Southern blot analysis of wild type (+/+) andrecombinant (+/−) ES cells, as well as of progeny of a heterozygotecross. Genomic DNA was digested with either EcoRI or BamHI andhybridized with probe 1. c, Northern blot analysis of mRNA (2 μg/lane)isolated from SHIP2^(+/+), SHIP2^(+/−) and SHIP2^(−/−) MEFs hybridizedwith a mouse SHIP2 or actin cDNA fragment as probes. d, Western blotanalysis of proteins (100 μg/lane) isolated from SHIP2^(+/+),SHIP2^(+/−) and SHIP2^(−/− MEFs probed with a rabbit anti-SHIP)2antibody. A single signal at 160 KDa is observed.)

[0033]FIG. 2 represents the impaired glucose homeostasis in SHIP2^(−/−)newborns. (a, Blood glucose (top) and plasma insulin (bottom)concentrations in 10 to 15 hour-old SHIP2^(+/+), SHIP2^(+/−) andSHIP2^(−/−) newborns injected or not within the first postnatal hourwith anti-insulin Ab ({fraction (1/800)} in 0.9% NaCl, 100 μl pernewborn). Values are expressed as the mean±SEM obtained from theanalysis of 9 SHIP2^(+/+), 8 SHIP2^(+/−), 8 SHIP2^(−/−), and 6anti-insulin Ab-treated SHIP2^(−/−) mice. b, Mortality of SHIP2^(−/−)newborns (n=22/group) injected or not with either D-glucose (7%, 100 μlper injection over the first postnatal day) or a neutralizing guinea piganti-insulin Ab ({fraction (1/800)} in 0.9% NaCl, 100 μl within thefirst postnatal hour). Mann-Whitney tests indicated that mortality ofnon-treated and treated SHIP2^(−/−) mice was significantly different(P<0.001). Mortality of SHIP2^(−/−) newborns treated with either normalguinea pig serum ({fraction (1/800)} in 0.9% NaCl, 100 μl per newborn)or 0.9% NaCl was not significantly different from non-treatedSHIP2^(−/−) 0 mice (data not shown). c, Northern blot analysis of PEPCK,TAT-5′ and G-6-Pase expression in liver of 2 to 3.5 hour-old SHIP2^(+/+)and SHIP2^(−/−) newborns treated or not with a single injection ofguinea pig anti-insulin Ab within the first hour after birth. The blotwas hybridized with a 18S RNA antisense oligonucleotide probe to controlfor equal loading of total RNA.)

[0034]FIG. 3 is showing the increased insulin sensitivity in adultSHIP2^(+/−) mice. (a, Blood glucose (top) and plasma insulin (bottom)concentrations in a glucose tolerance test. Values are expressed as themean±SEM obtained from 6 SHIP2^(+/+) (filled circles) and SHIP2^(+/−)(filled squares) mice that have indistinguishable body weight. *P<0.05;**P<0.01 (Student t test). b, Blood glucose concentrations in an insulintolerance test. Values are expressed as the mean±SEM obtained from 10SHIP2^(+/+) (filled circles) and SHIP2^(+/−) (filled squares) mice.**P<0.01; ***P<0.001 (Student t test). c, Northern and Western blotanalysis of total RNA and proteins isolated from skeletal muscles of 6-8week-old SHIP2^(+/+) and SHIP2^(+/−) mice. The Northern blot washybridized with a mouse SHIP2 cDNA fragment or a 18S RNA antisenseoligonucleotide as probes; an anti-SHIP2 Ab was used to detect SHIP2protein. d, GLUT4 expression in skeletal muscles from adult SHIP2^(+/+)and SHIP2^(+/−) mice. Mice were overnight fasted and injected (insulin)or not (basal) with insulin. The plasma membrane-rich fraction wasisolated from myocytes, and GLUT4 levels in this fraction weredetermined by Western blotting. The results are representative of 3separate experiments. The amount of GLUT4 was similar in the totallysate prepared from SHIP2^(+/+) and SHIP2^(+/−) myocytes (data notshown). e, Glycogen synthesis in isolated soleus muscles from adultSHIP2^(+/+) and SHIP2^(+/−) mice. Soleus muscles were isolated fromovernight fasted male SHIP2^(+/+) and SHIP2^(+/−) mice, and incubatedfor 60 min without or with (0.1-50 nM) insulin and [³H]-3-Glucose (5 mM,1 μCi/ml). At the end of the incubation, muscles were dissolved andglycogen was precipitated and counted. Left panel: Values are expressedas nmol glucose incorporated into glycogen per mg muscle protein and arepresented as means±SEM of 7-8 muscles (except at 0.1 nM where 4 muscleswere used). * P<0.02, ** P<0.03 (Student t test). Right panel: Resultsare expressed as percent of maximal insulin effect (determined bydividing the increment due to insulin at each hormone concentration bythe maximal increment in insulin effect at 50 nM).)

DETAILED DESCRIPTION OF THE INVENTION

[0035] In order to characterize SHIP2 molecule functions in vivo, theinventors have generated and analysed a mouse strain deficient from theSHIP2 gene according to the following preferred method.

[0036] Production of SHIP2 Deficient Mice and Genotyping

[0037] The targeting vector was constructed by replacing a 7.3 kbgenomic fragment containing exons 19-29 and the polyadenylation signalof the mouse SHIP2 gene by a neomycine resistance cassette (Schurmans etal. (1999)). The cassette was flanked by a 4.0 kb and a 5.5 kb mousegenomic DNA fragments at the 5′ and 3′ regions, respectively. R1 EScells were electroporated with the targeting plasmid linearized by SalI.Homologous recombination at the SHIP2 locus was confirmed by Southernblotting, and SHIP2^(+/−) ES cells were aggregated with morulae derivedfrom CD1 mice. The resulting chimaeric mice transmitted the mutantallele to the progeny. For genotyping, Southern analysis was performedwith specific probes (described in FIG. 1a) or by polymerase chainreaction using specific primers to amplify the neo gene and a specificexon deleted in the mutant allele.

[0038] Northern and Western Analysis

[0039] Messenger RNA was extracted from mouse embryonic fibroblasts(MEF) using a FastTrack kit (Invitrogen), and total RNA was purifiedfrom newborn liver and from adult skeletal and cardiac muscles using theRNeasy Mini Kit (Qiagen). 2 μg/lane of mRNA or 20 μg/lane of total RNAwere loaded on a 1% agarose gel and transferred to a nylon membrane. Themembranes were hybridized with mouse cDNA fragments coding for SHIP2,TAT-5′, C/EBP□, C/EBP□, aldolase B, actin or G-6-Pase as probes.Antisense oligonucleotide probes were used for PEPCK mRNA(5′-CAGACCATTATGCAGCTGAGGAGGCATT-3′) and 18S RNA(5′-GTGCGTACTTAGACATGCATG-3′) detection (Lee et al. (1997)).Supernatants of MEF homogenates isolated from SHIP2^(+/+), SHIP2^(+/−)and SHIP2^(−/−) day 13.5 embryos were analyzed by Western blot. Proteins(100 μg/lane) were separated by SDS-PAGE and transferred tonitrocellulose sheets. Saturation, incubation with rabbit anti-SHIP2antiserum and ECL detection were performed as described (Bruyns et al.(1999)). For in vivo GLUT4 expression, SHIP2^(+/+) and SHIP2^(+/−) mice(6-10 week-old) were overnight fasted, anaesthetized and intravenouslyinjected or not with 1 mU/g (body weight) insulin. After 5 min, theskeletal muscles from the hind limbs were removed. A plasmamembrane-rich fraction and a total lysate were prepared from myocytes asdescribed (Simpson et al. (1983), Higaki et al. (1999)). Aliquots ofproteins (100 μg) were separated by SDS-PAGE and blotted with anti-GLUT4antibody. Immune complexes were detected by using ¹²⁵I protein-A.

[0040] Glucose and Insulin Tolerance Tests

[0041] SHIP2^(+/+) and SHIP2^(+/−) mice (6-10 week-old) were fasted formore than 12 h and intraperitoneally injected with either 1.5 mg/g (bodyweight) D-glucose or 1 mU/g (body weight) insulin (Actrapid™, NovoNordisc, Denmark). Blood samples were drawn from the retro-orbital sinusat different time points. Blood glucose concentration was determinedenzymatically. Plasma insulin levels were determined using a rat insulinELISA kit™ (Mercodia AB, Uppsala, Sweden).

[0042] In Vivo Treatments with Glucose or Anti-Insulin Antibodies

[0043] Newborns received either repeated intraperitoneal injections ofD-glucose (7%, 100 μl per injection) during the first 24 hours afterbirth, or a single intraperitoneal injection of a guinea piganti-porcine insulin polyclonal Ab ({fraction (1/800)} in 0.9% NaCl, 100μl; ref AB1295™, Chemicon Int Inc, Temecula, Calif.) within the firstpostnatal hour. The neutralizing effect of this anti-insulin antibodywas demonstrated after injection in adult mice loaded with glucose:significantly increased blood glucose levels were observed after 30 and60 min, as compared to non-treated or normal guinea pig serum-treatedmice.

[0044] Output of Insulin from Pancreatic Islet Cells

[0045] Pancreatic islets (Malaisse et al. (1984)), prepared by thecollagenase procedure from the pancreas of 3-4 mice, were incubated ingroups of 8 islets each for 90 min at 37° C. in 1.0 ml of a Hepes- andbicarbonate-buffered medium containing 5 mg/ml bovine serum albumin and,as required, 11.1 mM D-glucose. The insulin released in the medium wasmeasured by radioimmunoassay.

[0046] Analysis of Glycogen Synthesis in Isolated Soleus Muscles

[0047] The 2 soleus muscles were rapidly isolated from overnight fastedSHIP2^(+/+) or SHIP2^(+/−) mice, and tied to stainless steel clips bythe tendons (Stenbit et al. (1996)). All incubations were carried out at37° C. under an atmosphere of 95% 02:5% CO₂ in 1 ml of Krebs-Ringerbicarbonate buffer (pH 7.35) supplemented with 1% bovine serum albumin(Fraction V, pH 7, Intergen) and 2 mM pyruvate. Following a 15 minpreincubation without insulin, muscles were incubated for 60 min withoutor with the indicated concentrations of insulin in the same mediumwithout pyruvate but with 3-[³H]-glucose (5 mM, 1 μCi/ml). Uponcompletion, muscles were dissolved in 1N NaOH, and aliquots of thealkaline solution were spotted onto Whatmann papers. Papers were droppedinto ice-cold 60% ethanol, and washed extensively (three washes of 20min each) in 60% ethanol before counting. All results were expressed permg muscle protein (determined by the well-known Pierce assay).

[0048] Results

[0049] After electroporation of embryonic stem (ES) cells with thetargeting vector, the inventors have used Southern blotting and twodifferent probes and restriction enzymes in order to identifyrecombinant clones with a 7.3 kb genomic DNA deletion on one allele ofthe SHIP2 gene (FIG. 1a, b). Crossing of chimeric males with C57BL/6females resulted in F1 heterozygous (SHIP2^(+/−)) mice that have noobvious abnormalities. The body weight at 8 weeks, life expectancy andspontaneous tumor incidence were not significantly different fromwild-type SHIP2^(+/+) mice. F1 SHIP2^(+/−) mice were then intercrossedand 182 viable offspring were genotyped at birth; of these, 47 (26%)were SHIP2^(+/+), 94 (51%) SHIP2^(+/−) and 41 (22%) SHIP2^(−/−). Thesefrequencies were within Mendelian expectations for transmission of anautosomal gene, and suggest that disruption of both SHIP2 alleles doesnot cause embryonic lethality. Day 13.5 embryo-derived mouse embryonicfibroblasts (MEF) were analyzed to confirm the reduced levels and thecomplete absence of SHIP2 mRNA (FIG. 1c) or protein (FIG. 1d) inSHIP2^(+/−) and SHIP2^(−/−) mice, respectively.

[0050] At birth, SHIP2^(−/−) mice were phenotypically indistinguishablefrom their littermates; most of them were able to feed, althoughprogressively less efficiently than SHIP2^(+/+) or SHIP2^(+/−) mice.Close monitoring revealed that SHIP2^(−/−) mice became progressivelycyanotic or pale and lethargic within the first 24 hours of life. Somenewborn SHIP2^(−/−) pups presented signs of respiratory distress and allfailed to gain weight (SHIP2^(+/+): 1.60±0.02 g, SHIP2^(+/−): 1.63±0.04g and SHIP2^(−/−): 1.20±0.01 g for a typical litter of 10 newborns, 18hours after birth; Student t test, P<0.005), and died within 3 daysafter birth. The cause of the respiratory distress observed in some ofthe SHIP2^(−/−) mice was not a lack of surfactant or an abnormaldifferentiation of surfactant-producing cells, since the expression ofsurfactant-associated proteins (SP-A, SP-B and SP-C) and of TTF-1 orC/EBP transcription factors was normal in the lungs of SHIP2^(−/−) mice.Moreover, hematoxylin and eosin staining of SHIP2^(−/−) lung, as well asof brain, heart, thymus, liver, stomach, pancreas, kidney, skin, muscle,spleen, bladder, and small and large intestines sections revealed noparticular abnormalities.

[0051] In newborns, severe hypoglycaemia is often associated withcyanotic episodes, apnea, respiratory distress, refusal to feed andsomnolence. No difference in blood glucose concentrations among thedifferent genotypes was observed 2 and 8 hours after delivery. However,when analyzed between 10 to 15 hours postpartum, blood glucoseconcentration in SHIP2^(−/−) mice was significantly lower than inSHIP2^(+/−) and SHIP2^(+/+) mice (FIG. 2a; Student t test, P<10⁻⁶).Hypoglycaemia was not due to glycosuria nor to an excessive secretion ofinsulin by the pancreatic beta cells: plasma insulin levels weresignificantly lower in SHIP2^(−/−) mice than in SHIP2^(+/−) andSHIP2^(+/+) mice (FIG. 2a; Student t test, P<0.05). Histology andimmunohistochemistry of pancreatic islets using anti-insulin, -glucagonand -somatostatin antibodies did not reveal any abnormalities of antigenexpression and distribution in SHIP2^(−/−) mice. To test whether thehypoglycaemia was the cause of postnatal death, SHIP2^(−/−) mice wereinjected with D-glucose. Hypoglycaemic SHIP2^(−/−) mice were transientlyrescued for up to 96 hours by repeated injections of D-glucose duringthe first 24 hours postpartum (FIG. 2b). About ten minutes after eachinjection, SHIP2^(−/−) newborns recovered a normal skin colour and wereless lethargic than non-injected mice. Prolonged survival was alsoobserved when SHIP2^(−/−) -mice were injected with a neutralizingantibody to insulin within the first hour after birth (FIG. 2b). Inaddition, anti-Insulin Ab injection of SHIP2^(−/−) mice significantlyincreased glucose concentrations at 10-15 hours postpartum as comparedto non-injected mice (FIG. 2a, Student t test, P<0.0002). These dataindicate that hypoglycaemia in SHIP2^(−/−) mice is not caused by anincreased production of insulin or decreased glucagon levels, but ratherresults from an increased sensitivity to insulin.

[0052] The liver plays a major role in glucose homeostasis at birth: itprovides glucose to the blood via gluconeogenesis. During that criticalperiod, transcription of several hepatic enzymes involved ingluconeogenesis is activated in response to hormonal and dietaryconditions. Interfering with glucagon or insulin signalling cascades ator just before birth results in delayed or premature appearance of theseenzymes, and in neonatal hypoglycaemia or diabetes. Expression ofphosphoenolpyruvate carboxykinase (PEPCK), a key enzyme ofgluconeogenesis, was very low or absent in liver of SHIP2^(−/−) mice, ascompared to SHIP2^(+/+) mice (FIG. 2c). Injection of SHIP2^(−/−) micewith a neutralizing anti-Insulin Ab restored a normal expression ofPEPCK mRNA (FIG. 2c). Expression of hepatic tyrosine aminotransferase(TAT-5′) and glucose-6-phosphatase (G-6-Pase), two other gluconeogenicenzymes also induced after birth, were also decreased, albeit to alesser extend than PEPCK (FIG. 2c). Levels of C/EBP, C/EBP and aldolaseB mRNA were unaffected by the mutation. Thus, the absence of SHIP2 leadsto decreased expression of several key gluconeogenic enzymes,contributing to hypoglycaemia in newborn SHIP2^(−/−) mice. Importantly,despite the low insulin levels found in mutant mice, the expression ofPEPCK was induced when insulin is neutralized early after birth. Takentogether, these data indicate that SHIP2^(−/−) liver cells have enhancedsensitivity to insulin in vivo.

[0053] Since SHIP2 is a critical negative regulator of insulinsensitivity in vivo, and since loss of SHIP2 leads to lethalhypoglycaemia in newborn mice, it was investigated whether decreasedamounts of SHIP2 expression in SHIP2^(+/−) mice (FIG. 1c, 1 d, 3 c)would alter insulin sensitivity. There was no significant difference inbasal blood glucose or plasma insulin levels between adult SHIP2^(+/+)and SHIP2^(+/−) mice, either after an overnight fasting (FIG. 3a), orwhen freely fed (glucose levels in freely fed mice: 9.8±0.5 mM versus8.8±0.2 mM in SHIP2^(+/+) and SHIP2^(+/−) mice, respectively; insulinlevels in freely fed mice: 1.17±0.29 μg/l versus 0.96÷0.16 μg/l inSHIP2^(+/+) and SHIP2^(+/−) mice, respectively). However, injection ofD-glucose resulted in a more rapid glucose clearance in SHIP2^(+/−) thanin SHIP2^(+/+) mice: glycaemia was significantly lower at all timepoints in SHIP2^(+/−) mice (FIG. 3a). The increased glucose clearance inSHIP2^(+/−) mice was not a consequence of an increased release ofinsulin: thirty minutes after glucose administration, insulin levelswere also significantly lower in SHIP2^(+/−) than in wild-type mice(FIG. 3a). Moreover, the output of insulin from isolated pancreaticislets in response to glucose was not significantly different inSHIP2^(+/+) and SHIP2^(+/−) mice. Insulin hypersensitivity wasdemonstrated when mice were injected with insulin, that is, asignificantly more profound hypoglycaemia was observed by 30 and 60 minafter injection in SHIP2^(+/−) mice than in SHIP2^(+/+) mice (FIG. 3b).

[0054] Insulin stimulates glucose transport and storage of glucose asglycogen into skeletal muscles through the translocation of the GLUT4glucose transporter from intracellular stores to the cell surface (Czechet al. (1999)), and glycogen synthase activation. Loss of one SHIP2allele resulted in reduced SHIP2 mRNA and protein expression in skeletalmuscle cells (FIG. 3c). After an overnight fasting, the amount of GLUT4transporter in the myocyte plasma membrane fraction was low but similarin SHIP2^(+/+) and SHIP2^(+/−) mice (FIG. 3d). However, when SHIP2^(+/+)and SHIP2^(+/−) mice were loaded with insulin, plasma membrane GLUT4levels were higher in SHIP2^(+/−) skeletal muscles, consistent with theincreased glucose clearance found in these mice. Glycogen synthesis inresponse to insulin stimulation, which reflects both glucose uptake andglycogen synthase activation, was analyzed in isolated soleus musclesfrom SHIP2^(+/−) and SHIP2^(+/+) mice (FIG. 3e). In basal condition orin the presence of maximally stimulating insulin concentrations (2 and50 nM), a similar glycogen synthesis was observed in SHIP2^(+/+) andSHIP2^(+/−) muscles. However, stimulation with lower, physiologicinsulin concentrations (0.1 or 0.3 nM) resulted in significantly higherglycogen synthesis in SHIP2^(+/−) muscles than in SHIP2^(+/+) muscles(FIG. 3e). When expressed in percent of maximal insulin effect, theinsulin dose response curve was shifted towards the lower insulinconcentrations in SHIP2^(+/−) mice, as compared to SHIP2^(+/+) mice(FIG. 3e). Together, data indicate that insulin sensitivity issignificantly increased in skeletal muscles from heterozygous miceexpressing only reduced amount of SHIP2 protein.

[0055] The incidence of adult onset diabetes mellitus has dramaticallyincreased and the disease is a major health care problem. Resistance tothe stimulatory effect of insulin on glucose utilization is a keypathogenic feature of most forms of adult onset (type II, or non-insulindependent) diabetes, and contributes to the morbidity associated withautoimmune (type I, or insulin-dependent) diabetes. It is crucial tobetter understand the molecular mechanisms that regulate insulinsignaling. Data in genetically modified mice identify SHIP2 as acritical and essential negative regulator of insulin signaling andinsulin sensitivity in vivo. Thus, SHIP2 is a novel therapeutic targetfor the treatment of type II diabetes, and a candidate gene predisposingto the same disease.

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1 4 1 1258 PRT Homo sapiens SHIP2 (1)..(1258) amino acid sequence ofhuman Type II SH2-domain-containing inosit ol polyphosphate5-phosphatase or SHIP 1 Met Ala Ser Ala Cys Gly Ala Pro Gly Pro Gly GlyAla Leu Gly Ser 1 5 10 15 Gln Ala Pro Ser Trp Tyr His Arg Asp Leu SerArg Ala Ala Ala Glu 20 25 30 Glu Leu Leu Ala Arg Ala Gly Arg Asp Gly SerPhe Leu Val Arg Asp 35 40 45 Ser Glu Ser Val Ala Gly Ala Phe Ala Leu CysVal Leu Tyr Gln Lys 50 55 60 His Val His Thr Tyr Arg Ile Leu Pro Asp GlyGlu Asp Phe Leu Ala 65 70 75 80 Val Gln Thr Ser Gln Gly Val Pro Val ArgArg Phe Gln Thr Leu Gly 85 90 95 Glu Leu Ile Gly Leu Tyr Ala Gln Pro AsnGln Gly Leu Val Cys Ala 100 105 110 Leu Leu Leu Pro Val Glu Gly Glu ArgGlu Pro Asp Pro Pro Asp Asp 115 120 125 Arg Asp Ala Ser Asp Gly Glu AspGlu Lys Pro Pro Leu Pro Pro Arg 130 135 140 Ser Gly Ser Thr Ser Ile SerAla Pro Thr Gly Pro Ser Ser Pro Leu 145 150 155 160 Pro Ala Pro Glu ThrPro Thr Ala Pro Ala Ala Glu Ser Ala Pro Asn 165 170 175 Gly Leu Ser ThrVal Ser His Asp Tyr Leu Lys Gly Ser Tyr Gly Leu 180 185 190 Asp Leu GluAla Val Arg Gly Gly Ala Ser His Leu Pro His Leu Thr 195 200 205 Arg ThrLeu Ala Thr Ser Cys Arg Arg Leu His Ser Glu Val Asp Lys 210 215 220 ValLeu Ser Gly Leu Glu Ile Leu Ser Lys Val Phe Asp Gln Gln Ser 225 230 235240 Ser Pro Met Val Thr Arg Leu Leu Gln Gln Gln Asn Leu Pro Gln Thr 245250 255 Gly Glu Gln Glu Leu Glu Ser Leu Val Leu Lys Leu Ser Val Leu Lys260 265 270 Asp Phe Leu Ser Gly Ile Gln Lys Lys Ala Leu Lys Ala Leu GlnAsp 275 280 285 Met Ser Ser Thr Ala Pro Pro Ala Pro Gln Pro Ser Thr ArgLys Ala 290 295 300 Lys Thr Ile Pro Val Gln Ala Phe Glu Val Lys Leu AspVal Thr Leu 305 310 315 320 Gly Asp Leu Thr Lys Ile Gly Lys Ser Gln LysPhe Thr Leu Ser Val 325 330 335 Asp Val Glu Gly Gly Arg Leu Val Leu LeuArg Arg Gln Arg Asp Ser 340 345 350 Gln Glu Asp Trp Thr Thr Phe Thr HisAsp Arg Ile Arg Gln Leu Ile 355 360 365 Lys Ser Gln Arg Val Gln Asn LysLeu Gly Val Val Phe Glu Lys Glu 370 375 380 Lys Asp Arg Thr Gln Arg LysAsp Phe Ile Phe Val Ser Ala Arg Lys 385 390 395 400 Arg Glu Ala Phe CysGln Leu Leu Gln Leu Met Lys Asn Lys His Ser 405 410 415 Lys Gln Asp GluPro Asp Met Ile Ser Val Phe Ile Gly Thr Trp Asn 420 425 430 Met Gly SerVal Pro Pro Pro Lys Asn Val Thr Ser Trp Phe Thr Ser 435 440 445 Lys GlyLeu Gly Lys Thr Leu Asp Glu Val Thr Val Thr Ile Pro His 450 455 460 AspIle Tyr Val Phe Gly Thr Gln Glu Asn Ser Val Gly Asp Arg Glu 465 470 475480 Trp Leu Asp Leu Leu Arg Gly Gly Leu Lys Glu Leu Thr Asp Leu Asp 485490 495 Tyr Arg Pro Ile Ala Met Gln Ser Leu Trp Asn Ile Lys Val Ala Val500 505 510 Leu Val Lys Pro Glu His Glu Asn Arg Ile Ser His Val Ser ThrSer 515 520 525 Ser Val Lys Thr Gly Ile Ala Asn Thr Leu Gly Asn Lys GlyAla Val 530 535 540 Gly Val Ser Phe Met Phe Asn Gly Thr Ser Phe Gly PheVal Asn Cys 545 550 555 560 His Leu Thr Ser Gly Asn Glu Lys Thr Ala ArgArg Asn Gln Asn Tyr 565 570 575 Leu Asp Ile Leu Arg Leu Leu Ser Leu GlyAsp Arg Gln Leu Asn Ala 580 585 590 Phe Asp Ile Ser Leu Arg Phe Thr HisLeu Phe Trp Phe Gly Asp Leu 595 600 605 Asn Tyr Arg Leu Asp Met Asp IleGln Glu Ile Leu Asn Tyr Ile Ser 610 615 620 Arg Lys Glu Phe Glu Pro LeuLeu Arg Val Asp Gln Leu Asn Leu Glu 625 630 635 640 Arg Glu Lys His LysVal Phe Leu Arg Phe Ser Glu Glu Glu Ile Ser 645 650 655 Phe Pro Pro ThrTyr Arg Tyr Glu Arg Gly Ser Arg Asp Thr Tyr Ala 660 665 670 Trp His LysGln Lys Pro Thr Gly Val Arg Thr Asn Val Pro Ser Trp 675 680 685 Cys AspArg Ile Leu Trp Lys Ser Tyr Pro Glu Thr His Ile Ile Cys 690 695 700 AsnSer Tyr Gly Cys Thr Asp Asp Ile Val Thr Ser Asp His Ser Pro 705 710 715720 Val Phe Gly Thr Phe Glu Val Gly Val Thr Ser Gln Phe Ile Ser Lys 725730 735 Lys Gly Leu Ser Lys Thr Ser Asp Gln Ala Tyr Ile Glu Phe Glu Ser740 745 750 Ile Glu Ala Ile Val Lys Thr Ala Ser Arg Thr Lys Phe Phe IleGlu 755 760 765 Phe Tyr Ser Thr Cys Leu Glu Glu Tyr Lys Lys Ser Phe GluAsn Asp 770 775 780 Ala Gln Ser Ser Asp Asn Ile Asn Phe Leu Lys Val GlnTrp Ser Ser 785 790 795 800 Arg Gln Leu Pro Thr Leu Lys Pro Ile Leu AlaAsp Ile Glu Tyr Leu 805 810 815 Gln Asp Gln His Leu Leu Leu Thr Val LysSer Met Asp Gly Tyr Glu 820 825 830 Ser Tyr Gly Glu Cys Val Val Ala LeuLys Ser Met Ile Gly Ser Thr 835 840 845 Ala Gln Gln Phe Leu Thr Phe LeuSer His Arg Gly Glu Glu Thr Gly 850 855 860 Asn Ile Arg Gly Ser Met LysVal Arg Val Pro Thr Glu Arg Leu Gly 865 870 875 880 Thr Arg Glu Arg LeuTyr Glu Trp Ile Ser Ile Asp Lys Asp Glu Ala 885 890 895 Gly Ala Lys SerLys Ala Pro Ser Val Ser Arg Gly Ser Gln Glu Pro 900 905 910 Arg Ser GlySer Arg Lys Pro Ala Phe Thr Glu Ala Ser Cys Pro Leu 915 920 925 Ser ArgLeu Phe Glu Glu Pro Glu Lys Pro Pro Pro Thr Gly Arg Pro 930 935 940 ProAla Pro Pro Arg Ala Ala Pro Arg Glu Glu Pro Leu Thr Pro Arg 945 950 955960 Leu Lys Pro Glu Gly Ala Pro Glu Pro Glu Gly Val Ala Ala Pro Pro 965970 975 Pro Lys Asn Ser Phe Asn Asn Pro Ala Tyr Tyr Val Leu Glu Gly Val980 985 990 Pro His Gln Leu Leu Pro Pro Glu Pro Pro Ser Pro Ala Arg AlaPro 995 1000 1005 Val Pro Ser Ala Thr Lys Asn Lys Val Ala Ile Thr ValPro Ala 1010 1015 1020 Pro Gln Leu Gly His His Arg His Pro Arg Val GlyGlu Gly Ser 1025 1030 1035 Ser Ser Asp Glu Glu Ser Gly Gly Thr Leu ProPro Pro Asp Phe 1040 1045 1050 Pro Pro Pro Pro Leu Pro Asp Ser Ala IlePhe Leu Pro Pro Ser 1055 1060 1065 Leu Asp Pro Leu Pro Gly Pro Val ValArg Gly Arg Gly Gly Ala 1070 1075 1080 Glu Ala Arg Gly Pro Pro Pro ProLys Ala His Pro Arg Pro Pro 1085 1090 1095 Leu Pro Pro Gly Pro Ser ProAla Ser Thr Phe Leu Gly Glu Val 1100 1105 1110 Gly Ser Gly Asp Asp ArgSer Cys Ser Val Leu Gln Met Ala Lys 1115 1120 1125 Thr Leu Ser Glu ValAsp Tyr Ala Pro Ala Gly Pro Ala Arg Ser 1130 1135 1140 Ala Leu Leu ProGly Pro Leu Glu Leu Gln Pro Pro Arg Gly Leu 1145 1150 1155 Pro Ser AspTyr Gly Arg Pro Leu Ser Phe Pro Pro Pro Arg Ile 1160 1165 1170 Arg GluSer Ile Gln Glu Asp Leu Ala Glu Glu Ala Pro Cys Leu 1175 1180 1185 GlnGly Gly Arg Ala Ser Gly Leu Gly Glu Ala Gly Met Ser Ala 1190 1195 1200Trp Leu Arg Ala Ile Gly Leu Glu Arg Tyr Glu Glu Gly Leu Val 1205 12101215 His Asn Gly Trp Asp Asp Leu Glu Phe Leu Ser Asp Ile Thr Glu 12201225 1230 Glu Asp Leu Glu Glu Ala Gly Val Gln Asp Pro Ala His Lys Arg1235 1240 1245 Leu Leu Leu Asp Thr Leu Gln Leu Ser Lys 1250 1255 2 28DNA Artificial primer 2 cagaccatta tgcagctgag gaggcatt 28 3 21 DNAArtificial primer 3 gtgcgtactt agacatgcat g 21 4 10 PRT Homo sapiensshort amino acid sequence of SHIP-2 (1)..(10) 4 Arg Thr Asn Val Pro SerTrp Cys Asp Arg 1 5 10

1. A mutant protein of an inositol polyphosphate 5-phosphatase of SEQ ID No. 1, comprising one or more amino acid mutations at S687, C689, N684, D690, or R691 in the amino acid sequence of RTNVPSWCDR (amino acids 682-691 of SEQ ID NO. 1).
 2. The mutant protein of claim 1, wherein said mutant present homozygously causes severe hypoglycemia.
 3. The mutant protein of claim 1, wherein said mutant present heterozygously causes unpaired glucose tolerance.
 4. The mutant protein of claim 1, wherein said mutant protein has a dominant negative effect on the wild-type inositol polyphosphate 5-phosphatase of SEQ ID No.
 1. 5. The mutant protein of claim 1, wherein said mutant protein is active in its SH2 domain activity and/or proline-rich domain activity, said mutant protein competing for SH2 domain binding protein or proline-rich domain binding protein with a wild type inositol polyphosphate 5-phosphatase of SEQ ID No. 1
 6. The mutant protein of claim 1, wherein said mutation is in the proline-rich domain of an inositol polyphosphate 5-phosphatase of SEQ ID No.
 1. 7. The mutant protein of claim 1, wherein the phosphorylation on a tyrosine residue of said 5-phosphatase is inhibited in said mutant protein. 